Naphtol as b1 phospate

The ES R1 and EC F9 cells were fixed with 2% paraformaldehyde in phosphate-buffered saline, PBS, pH 7.0, within 15 min. ALP activity was detected after incubation in a solution containing 10 ml 0.02 M Tris-HCl buffer (pH 8.7), 1 mg Naphtol-AS-B1-phospate, and 5 mg fast red dye Texas Red (all from Sigma) at 37°C for 1 h.
For immunofluorescence analysis, cells fixed in 4% paraformaldehyde in PBS for 1 h were washed and permeabilized with 0.5% Triton X-100. Nonspecific reactions were blocked by 10% chicken serum (Gibco/Invitrogen). Primary rabbit anti-Oct4 and goat anti-Gata4 antibodies (Santa Cruz Biotechnology) were used at a dilution of 1 : 100. The cells were incubated in a solution of primary antibodies in PBS-Tween 20 at 4°C overnight. Secondary chicken anti-rabbit and donkey anti-goat antibodies conjugated with Alexa Fluor 594 and Alexa Fluor 488 (Molecular Probes) were diluted at 1 : 800 in a blocking buffer and applied to the cells for 4 h at room temperature. DAPI (Molecular Probes) was applied for 15 min for nuclear staining. The cells were mounted and examined under a Leica DMRXA2 fluorescence microscope (Leica Microsystems GmbH). For negative controls, the primary antibodies were omitted, and the same staining procedure was used.

The cells were fixed with 2% paraformaldehyde in PBS, pH 7.0, for 15 min and incubated in a solution containing 10 ml 0.02 M Tris-HCl buffer pH 8.7, 1 mg Naphtol-AS-B1-phospate and 5 mg Fast Red dye (all from Sigma-Aldrich) at 37°C for 1 h.